1. Field of the Invention
The present invention relates to an integrated microfluidic electrospray chip system and its analytical method for fast identification of proteins. This system integrates improved protein hydrolysis, solid-phase extraction mechanism, electrophoresis chip and mass spectrometry to provide a continuous protein identification process that produces highly accurate results.
2. Description of Related Art
Capillary electrophoresis (CE) is a kind of widely used analytical technique owing to its advantages of fast separation, minute sample injection volume, high sensitivity and convenient operation [R. Kuhn, S. Hof. Kuhn, Capillary Electrophoresis: Principles and Practice, 1993, Springer-Verlag, Berlin Heidellergg N. Y. (U.S.A.)]. Due to the rapid progression of biotechnology in recent years, analytical techniques developed from capillary electrophoresis have also been widely applied in DNA-based research [Roche, M. E.; Oda, R. P.; Landers, J. P. Biotechnology Progress, 1997, 13, 659-668]. As biochemical technology and semiconductor process technology advanced, Manz applied miniaturized capillary electrophoresis on microchip to undergo sample separation in 1992 [Manz. A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C. I. Paulus, A.; Ludi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258], and since then, pushed the electrophoretic separation technique to a field of higher technology—chip-based electrophoresis.
Chip-based electrophoresis is a highly efficient assay technique for trace amounts of species [K. Seiler, D. Jed Harrison, A. Manz. Analytical Chemistry 65 (1993), 1481]. There are, for example, reports about depositing purified samples (e.g. PCR (polymerase chain reaction) amplified DNA sequence, enzyme and substrate, antibody and antigen) on the sample channels of biochips for assay [N.-H. Chiem, D. J. Harrison, Electrophoresis (1998), 3040]; [N.-H. Chiem, D. J. Harrison, Clinical Chemistry 44 (3) 591].
Protein identification process is widely applied in drug development and analysis as well as screening of patient specimens. Its primary purpose is to identify unknown protein molecules. For example, flatbed electrophoresis can separate thousands of protein molecules, but cannot identify the proteins from the separated signals. The protein identification process involves the use of proteinase to hydrolyze the protein in the gel into many peptide molecules, which are subject to mass spectrometry to detect the type and sequence of amino acids in the peptide, and then matched against the protein sequence database to decipher the type and source of the protein.
In protein identification process, the first step is to undergo protein hydrolysis. Conventionally it is done by adding proteinase (trypsin) to the protein solution to obtain products of protein digestion after 8-24 hours under 37° C. But for protein reacts with the immobilized proteinase, the protein digested fragments (peptides) and proteinase could be well separated by washing so that the hydrolysis product is free of trypsin to increase the accuracy of a subsequent identification step.
After hydrolysis, the solution contained protein digested fragments must be desalted to have the salt in the buffer removed before being fed to the mass spectrometer for analysis. Usually peptide separation may be achieved by high performance liquid chromatography (HPLC) before detection by mass spectrometry. But the whole process is time consuming and does not allow multianalysis of sample after one injection, which increases the sample consumption and reduces the analysis accuracy.